The present invention relates to a process for the isolation of a novel oligospirostanoside from Asparagus racemosus and to the novel oligospirostanoside obtained by said process. The present invention particularly relates to a novel chemical entity oligospirostanoside (hereinafter referred to as Immunoside) and structurally constructed as 3-0-[xcex1-L-rhamnopyranosyl-(1xe2x86x922)-xcex1-L-rhamnopyranosyl-(1xe2x86x924)-0-xcex2-D-glucopyranosyl]-25(S)-5xcex1-spirostan-3xcex2-ol (on the basis of IR, 1H and 13Cxe2x80x94NMR Data (CPD, DEPT, HOMOCOR, HETCOR and COLOC), isolated from Asparagus racemosus and biologically evaluated as a potent immunomodulatory agent.
The present invention also relates to a pharmaceutical composition containing the novel oligospirostanoside and to a method for immunomodulation using said oligospirostanoside
The Ayurvedic crude drug, Shatavari comprises decorticated roots of Asparagus racemosus wild [Kanitkar, U. K., Dange, P. S. and Pendse, G. S. J. Res. Indian Med. 3 (1969) 123; Medicinal Plants of Indiavol. 1, ed. by Satyavati, G. V., Raina, M. K. and Sharma, M. Indian Council of Medical Research, New Delhi (1976) 101].
Phytochemical investigations of the plant Asparagus racemosus, have resulted in isolation and characterization of steroidal glycosides [Ravikumar, P. R., Soman, R., Chetty, G. L. Pandey, R. C. and Sukhdev, Indian J Chem. 26 B (1987) 1012, Kar, Deepak Kumar and Sen Sumitra, Cell Chromosome Res. 7 (1984) 10], a novel cage type pyrrolizidine alkaloid, asparaginine [Sekine, T., Fukasawa, N., Kashiwagi, Y., Ruangrungsi, N. and Murakoshi, I. Chem. Pharm. Bull. 42 (1994) 1360] and a 9,10-dihydrophenanthrene derivative [Sekine, T., Fukasawa, N., Murakoshi, I. and Ruangrungsi, N. Phytochemistry, 44 (1997) 763].
The main object of the present invention is to provide a novel oligospirostanoside.
Yet another object of the present invention is to provide a process for isolation of a novel oligospirostanoside from Asparagus racemosus. 
Another object of the invention is to provide and characterize a novel sarsasapogenin glycoside Immunoside, an oligospirostanoside isolated from aqueous extract of Asparagus racemosus, present in the range of 0.0023-0.0045% w/w in the dried plant material.
Accordingly, the present invention provides a novel oligospirostanoside of formula 1,3-0-[xcex1-L-rhamnopyranosyl-(1xe2x86x922)-xcex1-L-rhamnopyranosyl-(1xe2x86x924)-0-xcex2-D-glucopyranosyl]-25(S)-5xcex2-spirostan-3xcex2-ol of the formula 1 below: 
The present invention also relates to a process for isolation of immunoside, 3-rhamnopyranosyl-(1xe2x86x922)-xcex1-L-rhamnopyranosyl-(1xe2x86x924)-0-xcex2-D-glucopyranosyl]-25(S)-5xcex2-spirostan-3xcex2-ol of the formula 1 below which comprises: 
The present invention also relates to a pharmaceutical composition comprising an effective amount of the novel oligospirostanoside 3-0-[xcex1-L-rhamnopyranosyl-(1xe2x86x922)-xcex1-L-rhamnopyranosyl -(1xe2x86x924)-0-xcex2-D-glucopyranosyl]-25(S)5xcex2-spirostan-3xcex2-ol contained in a pharmaceutically acceptable carrier.
In one embodiment of the invention, the amount of 3-0-[xcex1-L-rhamnopyranosyl-(1xe2x86x922)-xcex1-L-rhamnopyranosyl-(1xe2x86x924)-0-xcex2-D-glucopyranosyl]-25(S)5xcex2-spirostan-3xcex2-ol is in the range of 0.006 to 0.0125 mg per kg of body weight of subject to be treated.
The present invention also provides a method for immunomodulation in a immune suppressed animal comprising administering a pharmaceutically effective amount of 3-0-[xcex1-L-rhamnopyranosyl -(1xe2x86x922)-xcex1-L-rhamnopyranosyl-(1xe2x86x924)-0-xcex2-D-glucopyranosyl]-25(S)5xcex2-spirostan-3xcex2-ol.
In one embodiment of the invention, the amount of 3-0-[xcex1-L-rhamnopyranosyl-(1xe2x86x922)-xcex1-L-rhamnopyranosyl-(1xe2x86x924)-0-xcex2-D-glucopyranosyl]-25(S)5xcex2-spirostan-3xcex2-ol administered to the said animal comprises 0.006 to 0.0125 mg per kilogram of body weight of the animal.
The invention also relates to use of 3-0-[xcex1-L-rhamnopyranosyl-(1xe2x86x922)-xcex1-L-rhamnopyranosyl-(1xe2x86x924)-0-xcex2-D-glucopyranosyl]-25(S)5xcex2-spirostan-3xcex2-ol for the preparation of a pharmaceutical composition for immunomodulation in animals.
The process for isolation of a novel oligospirostanoside of formula 1 which comprises extraction of dried and powdered roots with a polar solvent with or without prior extraction with EtOAc, subjecting aqueous sol. of the desolvented extract to partitioning with CHCl3, EtOAc, n-BuOH and resolution of n-BuOH extract residue to adsorption/gel permeation chromatography using isotropic or graded elution, reverse phase purification on Lichroprep RP-8 and repeated crystallizations from methanol or ethanol. Physical constants and spectral data (1Hxe2x80x94NMR, 13Cxe2x80x94NMR, MS and I R spectral data) are used for characterization of novel isolate. Corroboration of assigned structure is done by permethylation and hydrolysis to get aglycone and partially methylated sugars to confirm linkage site and sequence of sugar units.
Estimation of the compound in the dried plant material is carried out by HPTLC densitometer scanning (0. 0023-0.0045%). The compound of the invention can be used for authentication of immunomodulatory formulation from Asparagus racemosus. The novel oligospirostanoside was evaluated for immunomodulatory activity and the results are given in Table 1. The compound of the invention is obtained as a white amorphous powder, mp 275xc2x0 C., [xcex1]D21xe2x88x9290.2[C 0.5% pyridine], molecular composition C45H74O16 derived from FABMS, MS (M+Na)+ m/z 893, elemental analytical data and 13Cxe2x80x94NMR, CPD and DEPT spectral data. Immunoside responded positively to the Liebermann-Burchard Reaction [Liebermann, G. (1885) Ber. Deut. Chem. Ges. 18, 1804; Burchard, H. (1890) Chem. Zentbl. 1, 25], negatively to the Ehrlich test [Kiyosawa, S and Hutoh, M. (1968) Chem. Pharm. Bull. 16, 1162; Tschesche, R; Siedel, L., Sharma, S. C. and Wulff, G. (1972) Chemische Berichte, 105, 3397] and positively to Molisch""s test indicating it to be spirostanol glycoside.
The following examples for the process of extraction are given by way of illustrations and therefore should not be construed to limit the scope of present invention: